Salivary Gland Cancer Patient-DerivedXenografts Enable Characterization of CancerStem Cells and New Gene Events Associatedwith Tumor ProgressionStephen B. Keysar1, Justin R. Eagles1, Bettina Miller1, Brian C. Jackson1,Farshad N. Chowdhury2, Julie Reisinger1, Tugs-Saikhan Chimed1, Phuong N. Le1,John J. Morton1, Hilary L. Somerset3, Marileila Varella-Garcia1, Aik-Choon Tan1,4,John I. Song2, Daniel W. Bowles1, Mary E. Reyland5, and Antonio Jimeno1,2,6
Abstract
Purpose: Salivary gland cancers (SGC) frequently present withdistant metastases many years after diagnosis, suggesting a cancerstem cell (CSC) subpopulation that initiates late recurrences;however, current models are limited both in their availabilityand suitability to characterize these rare cells.
Experimental Design: Patient-derived xenografts (PDX) weregenerated by engrafting patient tissue onto nude mice from oneacinic cell carcinoma (AciCC), four adenoid cystic carcinoma(ACC), and three mucoepidermoid carcinoma (MEC) cases,which were derived from successive relapses from the same MECpatient. Patient and PDX samples were analyzed by RNA-seq andExome-seq. Sphere formation potential and in vivo tumorigenicitywas assessed by sorting for Aldefluor (ALDH) activity and CD44-expressing subpopulations.
Results: For successive MEC relapses we found a time-depen-dent increase in CSCs (ALDHþCD44high), increasing from 0.2%
to 4.5% (P¼0.033), but more importantly we observed anincrease in individual CSC sphere formation and tumorigenicpotential. A 50% increase inmutational burden was documentedin subsequent MEC tumors, and this was associated withincreased expression of tumor-promoting genes (MT1E, LGR5,and LEF1), decreased expression of tumor-suppressor genes(CDKN2B, SIK1, and TP53), andhigher expression of CSC-relatedproteins such as SOX2, MYC, and ALDH1A1. Finally, genomicanalyses identified a novelNFIB–MTFR2 fusion in an ACC tumorand confirmed previously reported fusions (NTRK3–ETV6 andMYB–NFIB).
Conclusions: Sequential MEC PDX models preserved keypatient features and enabled the identification of genetic eventsputatively contributing to increases in both CSC proportion andintrinsic tumorigenicity, which mirrored the patient's clinicalcourse. Clin Cancer Res; 24(12); 2935–43. �2018 AACR.
IntroductionSalivary gland cancer (SGC) afflicts approximately 4,000 adults
in the United States annually and includes several distinct histo-types, of which mucoepidermoid carcinoma (MEC) and adenoid
cystic carcinoma (ACC) are the most prevalent (1, 2). Preclinicalmodels of SGC are scarce due to the difficulty in establishingmodels (3). These already limited models are further diluted bydisease histotype (4) and cell line misidentification (5). Researchusing patient-derived xenografts (PDX) from more commonmalignancies has led to enhanced preclinical and translationalresearch (6). However, to date, only ACC PDX models have beenreported (7, 8), with no reports characterizing PDX models fromthe most frequent subtype, MEC.
Late recurrences (longer than 5 years after diagnosis) manifest-ing primarily as distant metastases occur in upwards of 26% and17% of ACC and MEC patients, respectively (9). This is particu-larly striking in ACC patients with a promising 5-year survival rateof 75% to 80%, which plummets to 35% at 10 years and to 10%by 20 years (10, 11). Recurrences after a decade or more suggestthe persistence of a tumor cell population that is senescent, canself-renew, and upon migration to distant sites can replicate themorphology and heterogeneity of the originating tumor; thesecharacteristics are consistent with the accepted definition of acancer stem cell (CSC) subpopulation (12).
Earlier studies inferred the presence of SGC CSCs and testedsurface markers (CD44, CD133, and ABCG2) and CSC-relatedfactors (SOX2, OCT4, and NANOG; refs. 13, 14); however, these
1Division of Medical Oncology, Department of Medicine, University of ColoradoDenver School of Medicine (UCDSOM), Denver, Colorado. 2Department ofOtolaryngology, UCDSOM, Denver, Colorado. 3Department of Pathology, UCD-SOM, Denver, Colorado. 4Department of Biostatistics and Informatics, Universityof Colorado School of Public Health, Denver, Colorado. 5Department of Cra-niofacial Biology, University of Colorado Denver School of Dental Medicine,Denver, Colorado. 6Gates Center for Regenerative Medicine, UCDSOM, Denver,Colorado.
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
S.B. Keysar and J.R. Eagles contributed equally to this article.
Corresponding Author: Antonio Jimeno, University of Colorado Denver,Anschutz Medical Campus, 12801 East 17th Avenue, MS8117, Room L18-8111,Aurora, CO 80045. Phone: 303-724-2478; Fax: 303-724-3892; E-mail:[email protected]
did not prospectively define CSCs by sphere formation or tumorinitiation (15). Aldefluor was the sole marker used to determinethat the ALDHþ populationwas tumorigenic in ACCPDXmodels(16), and ALDHþCD44high cells from MEC cells lines formedspheres and initiated tumors (10, 17). Using ACC PDX modelsand primary cultures, Panaccione and colleagues (18, 19) deter-mined that CD133þ CSCs were maintained through SOX10 andNotch signaling and high expression of CD44.
Here, we report the successful engraftment of the first threeknown PDXmodels of a high grade MEC (derived from the samepatient after successive relapses), as well as one acinic cell carci-noma (AciCC), and four ACC cases. We have characterized thesomatic mutational landscape, confirmed the presence of previ-ously reported gene fusion events, and identified a novel genefusion. We determined that the ALDHþCD44high phenotypedefines the CSC fraction across ACC and MEC tumors usingsphere formation and in vivo tumor initiation. The detailedanalysis of MEC models generated from successive relapses iden-tified an increase in mutation load, associated with increasedexpression of tumor promoting genes, and decreased transcrip-tion of tumor suppressor genes. Finally, in a landmark finding insolid tumor CSCbiology, we found a time and relapse-dependentincrease in both CSC frequency, as well as individual CSC sphereforming potential in vitro and tumorigenicity in vivo. The accu-mulation ofCSCs over time contributing to disease progression orcancer relapse is considered a key principle in CSC biology, butthis concept has been elusive and difficult to demonstrate in solidtumors.
Materials and MethodsGeneration of patient-derived xenografts of SGCs
The protocol for studies involving human subjectswas approvedby the Colorado Multiple Institutional Review Board (COMIRB#08-0552) in accordance with the Belmont Report and U.S. Com-monRule. Informedwritten consentwas obtained fromall patientswhose tissues were used for this study. The University of ColoradoInstitutional Animal Care and Use Committee (IACUC) approved
all experiments involving mice. PDX generation and characteriza-tion was previously reported (20).
FACS and CSC implantation in vivoProcessing of tumor tissue for sorting, analysis, and in vivo
implantation of CSCs was previously reported (21).
IHCImmunohistochemistry (IHC) analyses were performed as
Sample preparation, whole-exome sequencing, and RNA-seqDNAwas isolated from blood and tumor with a DNeasy Blood
& Tissue kit (Qiagen), whereas total RNAwas isolated from tumortissue using an RNeasy kit (Qiagen). Isolated DNA and RNA weresent to the University of Colorado Cancer Center Genomics andMicroarray Core, which performed library preparation, sequenc-ing, and FASTQ generation. Single-end sequencing of mRNA(Poly A) was performed on an Illumina HiSEQ instrument withread lengths of 100bp (Illumina). DNA underwent paired-endsequencing using Agilent Technologies SureSelect Human AllExon Versions 5 and 6 with read lengths of 150bp (Agilent, SantaClara).
Sequencing confirmation of gene fusionsDNA sequencing was conducted as previously described
(22), and new primer sets were developed for the fusions listedbelow.
FISH analysis of gene fusionsFISH analysis of gene fusions was previously described (6)
and is further described in detail in the SupplementaryMaterials.
Translational Relevance
Salivary gland cancers are an orphan disease, and we haveestablished the first patient-derived xenograft models ofmucoepidermoid carcinoma (MEC) from tissue collected dur-ing subsequent surgeries for the same individual. CSCs weredefined by Aldefluor activity- and CD44-positive subpopula-tions, as well as sphere formation and in vivo tumor initiation.The novel findings in solid tumor CSC biology were increasesin the CSC fraction (from 0.2% to 4.5%), as well as increasedindividual CSC sphere formation and in vivo tumorigenicity inthe three successive MEC relapses. A 50% increase in muta-tional burden occurred in subsequent MEC relapses, associ-ated with tumorigenic changes in tumor-promoting and -sup-pressing genes; expression of the CSC-related proteins SOX2,MYC, and ALDH1A1 increased with relapses. A novel NFIB–MTFR2 fusion in an ACC tumor was identified. The frequencyof cancer stem cells as well as their associated sphere formingpotential and tumorigenicity increased with disease progres-sion and accumulating mutational burden.
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Statistical analysisExperiments were compared with a two-group t test. Calcula-
tions were done using GraphPad Prism version 7.0 and SPSSversion 11. Data are represented graphically as mean �SEM.
Data and materials availabilityMaterials will be shared per the University of Colorado's Office
for Technology Transfer policies and Institutional Review Board.
ResultsThe success of SGCPDXgeneration correlateswithdisease stage
After securing patient informed consent, excess tumor tissuesamples from 12 SGC surgeries were implanted subcutaneouslyon the flanks of athymic nude mice, including 5 ACC, 4 MEC, 1salivary duct carcinoma (SDC), 1 AciCC, and 1 mammary ana-logue secretory carcinoma (MASC). Engraftment rates variedbetween histotypes resulting in PDX models of 3 MEC (75%),3 ACC (60%), and 1 AciCC (100%; Supplementary Table S1;Supplementary Table S2). The engraftment rate of relapsedtumors was higher than that of primary cases and were 63% and20%, respectively (Supplementary Table S1), whereas time toinitial engraftment and subsequent passaging for MEC (3), ACC(3) and a single AciCC required on average 170� 46, 401� 7, and
343 days to generate tumors (>1,500mm3) respectively (Sup-plementary Fig. S1A). Morphology (hematoxylin and eosin;H&E staining) and cytokeratin 5 (CK5) staining were conservedin PDX tumors when compared with the originating patienttissue (Fig. 1A).
The most unique PDX were three models (CUSG006, �007,�012) generated from the same patient with a high-grade MECfrom three successive surgeries of relapses occurring over 3 years(Fig. 1B). The time between surgeries was similar (�9-10months), but by the third operation the tumor had migratedoutside of the primary parotid bed site, invading into the deepneck space and around the carotid artery. The growth rate ofCUSG012 was nearly identical to CUSG006 and CUSG007 oncestably passaged in mice (Supplementary Fig. S1B).
SGCs are hypomutated tumors with limited oncogene overlapWhole-exome sequencing (WES) of nine tumors from 7
patients with paired normal samples determined the mutationburden for each case (Fig. 1C). Possibly damaging somatic muta-tions present in more than one sample are displayed in a co-mutation (coMut) plot (Fig. 1D), whereas all mutations arereported in Supplementary Table S3. We observed substantialoverlap in the three MEC cases and an approximately 50%increased mutation burden in the two later samples, CUSG007
Figure 1.
Exome-seq and RNA-seq analyses identifying somatic mutations and gene fusions in SGC patient samples and PDX models. A, CUSG PDX models recapitulatedthe morphology (H&E) and CK5 staining of the originating patient tumor. B, Three PDX models engrafted from successive surgeries by the same patient.The time between tissue collection of CUSG006 and CUSG007, as well as CUSG007 and CUSG012, was consistent at approximately 10 months. C, Mutationburden (somatic mutations scored as possibly damaging) for each SGC case analyzed by Exome-seq. D, CoMut plot of somatic mutations scored as possiblydamaging occurring in two ormore cases. Gray boxes, missensemutations; blue boxes, nonsensemutations, yellowboxes, frameshiftmutations. Bold cases are fromtissue collected from successive surgeries from the same patient. E, Identified NFIB–MTFR2 gene fusion product. From top to bottom: Chromosomal locations,exon numbers, Sanger sequencing, and protein product. F, CUSG004 patient normal tissue, patient tumor tissue (F0), and PDX tumor tissue (F1, F5)hybridized with the NFIB(SG)/MTFR2(SR) FISH fusion probe set. Patient tumor and PDX tissue showed a positive pattern. Tumor adjacent normal tissuecollected at the time of surgery was negative for the fusion product. Arrows indicate fused signals.
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(62 mutations) and CUSG012 (58 mutations), compared withCUSG006 (41 mutations). We found that CUSG012 hadacquired an inactivating mutation in the SHPRH gene. Thisgene has been shown to suppress genomic instability (23),which may allow for rapid accumulation of mutations andtumor progression. When filtered by known somatic cancer-related mutations (COSMIC), we observed few oncogenicmutations per tumor (0 mutations in CUSG002, CUSG003,CUSG006, CUSG007; 1 mutation in CUSG004, CUSG005,CUSG012; 2 mutations in CUSG013 [ATM, KDM6A]), exceptfor the SDC case (CUSG008; 5 COSMIC mutations), whichincluded missense mutations in ERBB2, CDH1, and BAP1(Supplementary Fig. S1B). We also identified inactivatingmutations in genes with putative roles as tumor suppressorsand promoting genomic stability, including missense muta-tions of RB1CC1 in multiple histotypes (MEC, SDC, andMASC), whereas SHPRH mutations were found in two cases(MEC and SDC). We found that two ACC tumors (CUSG004,�005) had frameshift mutations in SPEN and KMT2D.
Gene fusions are a dominant source of genomic damage insome cases of SGC
The analysis of SGC tumor RNA-seq data using the STAR-fusionpipeline identifiedmultiple high-confidence gene fusion events inpatient tumors. Because single-end sequencing was performed,junction read counts, or the number of reads that split alignmentbetween two genes, were used as fusion evidence (SupplementaryTable S4).We identified a novel fusion event,NFIB-MTFR2, in thehypomutated ACCmodel CUSG004. Thismolecular fusion eventwas confirmed by Sanger sequencing (Fig. 1E) and subsequentlyvisualized by FISH using green 5'NFIB and the red 3'MTFR2DNAprobes in the CUSG004 patient tumor, but not in a matchednormal tissue sample (Fig. 1F) or other controls (SupplementaryFig. S2; Supplementary Table S5). We identified several previ-ously reported fusions, including NTRK3-ETV6 (CUSG002,MASC) and MYB-NFIB (CUSG005, ACC; SupplementaryTable S4). Coinciding with its high mutation burden (Fig.1C), CUSG008 (SDC) had numerous genomic rearrangements,including ADNP2-KCNG2, YTHDF1-NKAIN4, and LAMA5-FTCD (Supplementary Table S4). Finally, the NFIB–MTFR2(CUSG004) and MYB–NFIB (CUSG005) fusions observed inpatient tumor was conserved following engraftment on mice.
SGC gene expression analysis overlaps in histologic subtypesWe first assessed unsupervised gene clustering and the drift in
gene expression following PDX engraftment on mice and subse-quent passaging by analyzing patient tissue and multiple PDXpassages for three cases by RNA-seq. We found that the two ACCcases, CUSG004,�005, clusteredmore closely than theMEC case,CUSG006 (Supplementary Fig. S3A). We also found that 85% ofgenes that had significant changes in expression following engraft-ment on nude mice overlapped across all three cases (Supple-mentary Fig. S3B–S3E) and were related to immune response(Supplementary Fig. S4A–S4D). Numerous differentiallyexpressed genes were identified when comparing ACC or MECsamples versus all others (Supplementary Table S6). Gene setenrichment analysis (GSEA) identified significant upregulation ofthe Hallmark Pathways, "MYC Targets," "Mitotic Spindle," and"E2F Targets" in ACC tumors (Fig. 2A).
GSEA comparing the PDX tumors engrafted from subsequentMEC relapses identified the upregulation of Hallmark pathways
over time, including "E2F Targets," "Myc Targets," "DNA Repair,"and "TGF-beta" and downregulation of "Epithelial to Mesenchy-mal Transition" and "Inflammatory Response" (Fig. 2B). Whencomparing tissue collected from thefirst two surgeries (CUSG006,CUSG007), we observed progressively increased expression ofgrowth promoting genes (CR1 [97-fold], MAGEC2 [21-fold],MMP1 [3.1-fold], and HEY1 [2.1-fold]). We next comparedtissue from the second and third surgeries and found expressionof genes related to migration (MT1E [1,445-fold]), survival (EN1[6.9-fold]) and CSCs (LGR5 [28-fold], LEF1 [19-fold]) to bedramatically enriched in the relapsed third tumor (Fig. 2C). Justas striking, expression of key tumor suppressors (CDKN2B[-1,628-fold], TP53 [-2.3-fold], SIK1 [-1,709-fold]) was alsoinhibited in this same case.
Next, we assessed gene-expression changes throughout diseaseprogression by examining protein levels in patient tissue by IHC.Tumor cell proliferation, Ki67 expression, didnot changebetweencases CUSG006, �007, and �012 (Fig. 2D). However, we didobserve incremental increases in pSMAD2 staining andALDH1A1-high cells by IHC (Fig. 2D). Changes in gene expres-sion and IHC were confirmed by western blot for key pathways,which were replicated three times with different PDX tumors.Levels of pSMAD2, pSMAD3, NOTCH1, HES1, SOX2, ALDH1A1,and MYC increased over disease progression in the three PDXcases, whereas EGFR signaling (EGFR, pEGFR, and pMAPK)decreased (Fig. 2E).
SGC subpopulations with sphere-forming potential share acommon phenotype
We determined Aldefluor activity and CD44 expression in twoACC and threeMECPDXmodels and found the ALDHþCD44high
population consistently fell between 0.15% and 4.5%, exceptfor the ACC PDX model CUSG005 that had a dual positivepopulation of 17% (Fig. 3A; Supplementary Table S7). Cellsfrom PDX tumors were sorted by Aldefluor activity andCD44 expression and seeded as spheres (5 � 103 cells/wellALDHþCD44high, ALDHþCD44low, ALDH�CD44high or 5 �104 cells/well ALDH�CD44low) in 96-well low-bind plates. TheALDHþCD44high population generated the most tumor spheresfor both ACC (CUSG004 P ¼ 0.032, CUSG005 P < 0.001) andMEC (CUSG007 P < 0.001, CUSG012 P ¼ 0.018) when sortedfrom PDX. The initial MEC tumor of three successive surgeries(CUSG006) was the sole exception, where no sorted subpop-ulation generated more than 3 spheres per well (Fig. 3B;Supplementary Fig. S5). Out of all subpopulations sortedacross cases, the CUSG012 ALDHþCD44high subpopulationconsistently formed the largest spheres across all cases andhistotypes (Fig. 3B).
The ALDHþCD44high subpopulation from the three MECrelapses increased from 0.2% (CUSG006) to 0.3% (CUSG007)and then to 4.5% (CUSG012; ref. Fig. 3C; Supplementary TableS7). As noted above, subpopulations sorted from CUSG006tumors had difficulty generating spheres, whereas CUSG007ALDHþCD44high cells readily formed spheres (Fig. 3C). Of note,all subpopulations sorted from CUSG012 tumors were able togenerate spheres (Fig. 3C and D); however, the ALDHþCD44high
cell subpopulation had the greatest sphere-generating potential,forming themost and the largest spheres across all three cases (Fig.3C). Comparing sortedALDHþCD44high andbulk tumor cells, wefound that HES1, SOX2, and pSMAD2 were enriched in sphere-forming ALDHþCD44high cells (Fig. 3D).
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Tumorigenic populations induce in vivo tumors in cell dilutionstudies
Tumor formation in mice using cell dilution assays remainsthe gold standard to identify CSCs (24, 25). All four tumorsubpopulations (102 and 103 cells for ALDHþCD44high,ALDHþCD44low, ALDH�CD44high; 105 cells for ALDH�C-D44low) and bulk tumor cells (104 and 105 cells) from oneACC (CUSG004) and three MEC (CUSG006, �007, �012)PDX were implanted into the flanks of nude mice and mon-itored for up to 500 days for tumor formation. TheALDHþCD44high subpopulation was the most tumorigenicwhen �103 cells were injected (Supplementary Table S8), buttumor initiation/growth took over a year when 10-fold fewer(�102) ALDHþCD44high cells were implanted (Fig. 4A), sug-gesting that CSCs can remain senescent for long periods beforeactively proliferating. Importantly, 103 ALDHþCD44high cellswere as tumorigenic as 105 bulk tumor cells supporting that it isthe approximately 1% CSC fraction within bulk cells that bearstumorigenicity; indeed, 105 ALDH�CD44low cells rarely formedtumors (Supplementary Table S8). Tumors resulting from CSCimplantation harbored CSC fractions similar to the originatingPDX (Supplementary Table S7), and subsequent passaging oftumor subpopulations sorted from CSC-generated tumors(CUSG004, CUSG007) gave similar results (SupplementaryTable S8 and Fig. 4B).
MECCSCs increase their fraction and individual tumorigenicityupon successive relapses
No subpopulations sorted from CUSG006 tumors generatedtumorswhereas ALDHþCD44high, and to a lesser extent ALDHþC-D44low cells, from CUSG007 and CUSG012 cells readily formedtumors in cell dilution studies with inoculates as low as 102 cells(Supplementary Table S8). It is important to note again that theALDHþCD44high fraction increased from0.3% to4.5%during theprogression of CUSG007 to �012, possibly accounting for thesubtly lower single marker (ALDH�CD44high and ALDHþC-D44low) subpopulation tumorigenicity in CUSG012.
DiscussionOrphan diseases like SGC are often understudied due to a
scarcity of laboratory models such as cell lines or PDX forfunctional, mechanistic, and/or therapeutic experiments. Cur-rently, research into the molecular drivers responsible for SGCinitiation and progression is limited, and this is perhaps reflectedin the lack of targeted therapies for this disease. There is alsoincomplete evidence that CSCs promote proliferation and relapseof SGC tumors across histotypes (16, 17). Therefore, establishingPDX models will greatly aid in the identification of key genomicand expression alterations in engrafted cases and in the definitionof the CSC subpopulations across SGC histotypes. This will
Figure 2.
Gene expression analysis of SGC patient tissue and PDX models. A, Hallmark gene sets enriched for both upregulation and downregulation in ACC versusall other SGC cases. B, Hallmark gene sets enriched for both upregulation and downregulation when comparing PDX models arising from successivesurgeries. CUSG006¼ 1st surgery, CUSG007¼ 2nd surgery, CUSG012¼ 3rd surgery. C, Waterfall plots denoting changes in gene expression of >Log2fold change (P < 0.05) between three PDX cases engrafted from successive surgeries. D, IHC staining of patient tumor tissue collected from the samepatient over consecutive surgeries showed pSMAD2 staining increased over time along with the number of highly positive ALDH1A1 cells (arrows).E, Western blot analysis of PDX tumor tissue highlights increased expression of CSC-related, Notch signaling, and TGF-b pathway proteins during tumorprogression.
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provide additional and valuable tools in determining the pro-cesses that lead to late recurrences and metastasis (9) and devel-oping hypothesis-based novel therapeutics (26). In this report, we
show the first successful establishment of high-grade MEC PDXmodels reported to date, the fact that these originated from thesame individual at different disease times, and the observation of
Figure 3.
Sphere formation by SGC PDX tumor subpopulations. A, Representative histograms of sorted ALDHþCD44high populations. B, Generation of spheres by tumorsubpopulations sorted from two ACC (CUSG004, �005) and three MEC (CUSG006, �007, �012) PDX models measured by Top spheres per well (�50 mm)andBottom sphere size. Gray shaded graphs are for PDX tumors generated from the same tumor over successive surgeries.C,ALDHþCD44high percentage, spheresper well, and sphere size generated by the ALDHþCD44high subpopulation sorted from PDX models that were engrafted from tissue collected from successivesurgeries in the same patient. D, CUSG012 ALDHþCD44high cells have increased SOX2, Hes1, and pSmad2 levels than bulk tumor cells; �, P < 0.05.
Figure 4.
Tumor initiation by SGC PDX tumor subpopulations. A, 103 ALDHþCD44high cells initiate tumors that proliferate as rapidly as 105 bulk tumor cells. When injectedinto the flanks of nude mice, as few as 102 CUSG004 ALDHþCD44high cells initiated tumors after not proliferating (forming detectable tumors) for nearly1 year. B, Tumors initiated by SGC CSCs (ALDHþCD44high) recapitulate the morphology (H&E) and CK5 staining for the originating patient and PDX tumors.
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mutation accumulation and enhanced growth-promoting genedysregulation over time. In addition, a novel NFIB--MTFR2 genefusion was identified in an ACC case. Importantly, we demon-strate that both CSC frequency and individual CSC "aggres-siveness" (sphere forming potential, tumorigenicity) increase insubsequent relapses.
The characterization of the genomic and expression changes ofsuccessive relapses of an increasingly aggressive tumor over timerevealed that during the approximately 10 months between thefirst (CUSG006) and second (CUSG007) relapses there weredramatic increases in CR1 and MAGEC2 expression, which reg-ulate the CSC compartment of colorectal tumors (27) and canpromote metastases through STAT3 (28), respectively. In com-paring CUSG007 and CUSG012, we found that CUSG012 hadacquired an inactivating mutation in SHPRH, a gene that sup-presses genomic instability (23), which could account for theaccompanying genomic and gene-expression changes. Alongwithdownregulation of tumor suppressors (CDKN2B, SIK1, TP53), wenoted increased expression of genes associated with survival(EN1; ref. 29), Wnt signaling (LEF1), and colon stem cells andcolorectal CSCs (LGR5). These gene-expression changes wereaccompanied by increased TGF-b signaling and expression ofknown protein effectors of the CSC phenotype (SOX2, MYC,ALDH1A1), suggesting a transition to a CSC-enriched state.
To formally characterize CSCs, we used both sphere formationand tumor initiation in mice using cell dilution assays, the in vitroand in vivo "gold standards" when prospectively defining CSCs(15). Similar to previous CSC studies in solid tumors (15) theALDHþCD44high fraction ranged between approximately 0.1%and 3% in four out of five cases. However, the ALDHþCD44high
population in the CUSG005 tumor was >15%, which may bedue to the homogeneity of this MYB-NFIB fusion-driven tumor(30). The ALDHþCD44high population consistently generatedthe most, and the largest, spheres compared with other subpo-pulations across ACC and MEC subtypes. As few as 102
ALDHþCD44high cells initiated tumors in vivo, and resultingtumors recapitulated the morphology, tumor cell populations,and CSC fraction as that of the originating tumor. Possibly themost striking data arose from the inherently controlled assess-ment of CSC profiles and properties from PDX models arisingfrom three subsequent relapses from the same subject. Increasedmutation load and ensuing gene dysregulation ultimately led toboth increased CSC fraction and individual CSC aggressiveness(sphere formation, tumorigenicity), in what we propose is arelevant observation highlighting the translational value of PDXmodels. This finding supports a core tenet in CSC biology.Similarly, it has been reported that CSC directly extracted frompre- and post-treatment samples of breast cancers showedincreases in breast CSC numbers and sphere formation potentialin response to cytotoxic therapy (31),emphasizing that originat-ing patient samples are critical to study delicate biological pro-cesses such as the characterization of the CSC fraction.
The timing of CSC initiated tumor formation closely followedwhat is observed in the clinic, as 100 highly tumorigenic CSCstook nearly 250 days to give rise to measurable (�100 mm3)tumors on mice, a size still several-fold smaller than what isclinically or radiologically detectable in patients. The ability ofa few cells to survive undetected for many months in a hostileenvironment before proliferating are characteristics consistentwith residual disease leading to late recurrence and metastasesin SGC patients (9). The observation that PDX-derived CSCs can
model the clinical behavior of SGC is critical to support theutilization of these models to study the process of metastasis andto identify relevant therapies.
We successfully engrafted PDX models for three MEC, threeACC, and one AciCC cases that generally preserved the originatortumor's morphology. The time required for initial SGC PDXengraftment following patient tumor implantation exceeded ayear in some cases, which was much longer than what we havepreviously observed in our extensive experience with head andneck squamous cell carcinomas (HNSCC). HNSCC commonlygrow within 3 to 4 months (22); notably, this follows the usualtiming of HNSCCs relapses that commonly occur in the first yearafter diagnosis (32). The growth dynamic of SGC PDX is alsoconsistent with the protracted natural history where SGC distantrelapses decades after diagnosis are common (9, 10) and previousSGC PDX reports (7, 8). To our knowledge, we have generated thefirst functional PDX models of MEC, which retain the morphol-ogy of their respective histotypes. An interesting observation isthat the majority of successfully engrafted cases were relapses,consistent with pancreatic and breast cancers (33, 34).
SGCs are hypomutated compared with other neoplasms (35).Through WES analysis, we determined that ACC tumors(CUSG004,CUSG005,CUSG013)hadbetween19 and25 somat-ic mutations, which is consistent with a previous study thatreported 2-35 mutations per ACC case (36). We did not observehigh levels of TP53mutations or alterations in PI3K signaling thatwere reported in studies using targeted NextGen Sequencing of182-315 cancer genes (37–39). Thismay be due in part to the lackof sequenced paired normal tissue to remove germline SNPs/mutations in these other reports. However, we did observe muta-tions in cancer associated genes that have been reported usingmultiple platforms. We found that two of three ACC tumorsharbored frameshift mutations in SPEN, a negative regulator ofNotch signaling, which ismutated in up to 20%of ACC cases (36,38). The third ACC case had mutations in both KDM6A and ATMthat aremutated in approximately 15% and approximately 5% ofACCs respectively (38, 40). The BRCA1-Associated Protein 1(BAP1) wasmutated in the single SDC case (CUSG008) analyzed,which is a gene consistentlymutated in 5% to 10%of SGCs acrossdisease histotypes (37, 38, 40). We also observed inactivatingmutations across histotypes in putative tumor-suppressorsRB1CC1, RARRES1 and TP53BP1 (41–43), as well as in SHPRH,which suppresses genomic instability (23) by promoting errorfree replication (44). Loss of SHPRH function may account forrapid changes in gene expression and progression of theCUSG012 tumor. Also, twoACCcaseswith lowmutationburdensshared damaging rearrangements in the tumor-suppressorKMT2D (45). Finally,we have confirmed thepresence of commonSGC genomic alterations and novel mutations that may allow fortumorigenesis and disease progression.
Given this low frequency of oncogenic mutations, we testedgene fusions known to occur in several SGC histotypes, includingETV6-NTRK3 fusions in MASCs and MYB–NFIB fusions in ACCs(46, 47), and these genetic events were identified in casesCUSG002 (MASC) and �005 (ACC), respectively. We identifiedanovel gene fusionbetween theNFIB andMTFR2 genes in anACCPDX model (CUSG004). This is likely a driver event because theMYB domain of the MYB–NFIB fusion promotes carcinogenesisthrough its role as a transcription factor (30). Notably, NFIB hasbeen identified as an oncogene in small-cell lung cancer where itappears to regulate cell proliferation and viability through
Salivary Cancer Stem Cells Increase with Disease Progression
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transcriptional regulation (48). Furthermore, the NFIB–MTFR2fusion results in the loss of the transcriptional regulatory domainof NFIB and leads to a truncated NFIB protein. Further studiesexamining the consequences of this truncation are merited asNFIB is thought to be important in the regulation of the TGF-betaand Sonic Hedgehog pathways (49).
In summary, here we report detailed genomic and CSC char-acterization from a comprehensive panel of SGC PDX, whichincludes three sequential models from the sameMEC patient thathave enabled us to dissect themolecular evolution and functionalchanges in CSCs over time. This is the first report of successfulMEC PDX generation. We observed a doubling in the mutationburden in subsequent relapses and found an enrichment of pro-survival gene expression and "stemness." The size of the CSCsubpopulation increased over time, and in addition the CSCs hada significant rise in sphere initiation capacity and in vivo tumor-igenicity. Overall these findings suggest that CSCs may be asso-ciated with tumor progression inMEC, as well as highlighting theimportance of SGC PDX as a valid tool in expanding our under-standing of themolecular progression of cancer and the impact ofdisease progression on tumorigenic cell subpopulations, such asCSCs.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: S.B. Keysar, M.E. Reyland, A. JimenoDevelopment of methodology: S.B. Keysar, J.R. Eagles, A.-C. Tan, A. Jimeno
Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.B. Keysar, J.R. Eagles, B. Miller, F.N. Chowdhury,J. Reisinger, T.-S. Chimed, J.J. Morton, M. Varella-Garcia, J.I. Song, D.W. Bowles,A. JimenoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.B. Keysar, J.R. Eagles, B. Miller, B.C. Jackson,H.L. Somerset, M. Varella-Garcia, A.-C. Tan, D.W. Bowles, A. JimenoWriting, review, and/or revision of the manuscript: S.B. Keysar, J.R. Eagles,B. Miller, F.N. Chowdhury, J. Reisinger, P.N. Le, J.J. Morton, H.L. Somerset,M. Varella-Garcia, J.I. Song, D.W. Bowles, M.E. Reyland, A. JimenoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. JimenoStudy supervision: A. Jimeno
AcknowledgmentsThis work was supported by NIH grants R01-CA149456 (to A. Jimeno), R21-
DE019712 (to A. Jimeno), R01-DE024371 (to A. Jimeno), P30-CA046934(University of Colorado Cancer Center Support Grant), R01-DE015648 (toM.E. Reyland), R56-DE023245 (to M.E. Reyland and A. Jimeno), the AdenoidCystic Carcinoma Research Foundation (to A. Jimeno and M.E. Reyland),University of ColoradoAdenoidCystic Cancer Research Fund (toD.W. Bowles),the Daniel and Janet Mordecai Foundation (to A. Jimeno), and the Peter andRhonda Grant Foundation (to A. Jimeno). The authors wish to thank thepatients who donated their tissue, blood and time, and to the clinical teamswho facilitated patient informed consent, as well as sample and dataacquisition.
The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received January 1, 2018; revised February 12, 2018; accepted March 14,2018; published first March 19, 2018.
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Salivary Cancer Stem Cells Increase with Disease Progression
2018;24:2935-2943. Published OnlineFirst March 19, 2018.Clin Cancer Res Stephen B. Keysar, Justin R. Eagles, Bettina Miller, et al. Associated with Tumor ProgressionCharacterization of Cancer Stem Cells and New Gene Events Salivary Gland Cancer Patient-Derived Xenografts Enable
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